Mineralization of cartilage and bone occurs by a series of physicochemical and biochemical processes that together facilitate the deposition of hydroxyapatite in specific areas of the extracellular matrix (ECM). Experimental evidence has pointed to the presence of hydroxyapatite (HA) crystals along collagen fibrils in the ECM and also within the lumen of chondroblast- and osteoblast-derived matrix vesicles (MVs). Our working model is that bone mineralization is first initiated within the lumen of MVs. In a second step, HA crystals grow beyond the confines of the MVs and become exposed to the extracellular milieu where they continue to propagate along collagen fibrils. Our recent data have indicated that tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role in restricting the concentration of extracellular inorganic pyrophosphate (PPi), a mineralization inhibitor, to maintain a Pi/PPj ratio permissive for normal bone mineralization. Using a variety of single and double gene knockout experiments we have found that mice deficient in TNAP function, i.e., Akp2-/- mice, display osteomalacia due to an arrest in the propagation of HA crystals outside the MVs caused by an increase in extracellular PPj concentrations. Inside the MVs, however, HA crystals are still present in Akp2-/- mice. We have also found that the co-expression of TNAP and type I collagen is necessary to cause mineralization of any ECM indicating that propagation of HA crystals in the bone ECM is intimately dependent on the presence of type I collagen. But why are Akp2-/- mice born with a mineralized skeleton and have HA crystals in their MVs? We hypothesize that a newly identified soluble phosphatase called PHOSPHO1, present in the MVs, is responsible for increasing the local concentration of Pi inside the MVs to change the Pi/PPi ratio to favor precipitation of HA seed crystals. We will test this hypothesis by affecting the first and second steps of MV-mediated mineralization using a genetic and pharmacological approach. Experimentally we will characterize the mineralization abnormalities and related metabolic changes in mice deficient in Phospho1 expression compared to Akp2-/- mice and assess the effect of the simultaneous inactivation of the Phospho1 and Akp2 genes on skeletal mineralization. We will also study the effects of ablating or inhibiting PHOSPHO1 and/or TNAP activity on the ability of osteoblast- derived MVs to initiate and propagate calcification in vitro. Our work will provide fundamental insights into the mechanisms of normal bone mineralization. Our project will also produce valuable tools and reagents that will facilitate future studies aimed at understanding the development of diverse bone mineralization and soft tissue ossification abnormalities that include some diseases of great public health concern, e.g., osteoarthritis, osteoporosis, and arterial calcification.